Triple-negative breast cancers [ER/PR-/-, HER2/neu wt, EGFR+] frequently occur in young women and carry a poor prognosis. While not all triple-negative breast cancers are lethal, triple-negative breast cancers have 86% 5-year mortality in pre-menopausal African American women. Since many triple-negative breast cancers are chemotherapy-resistant at diagnosis, there is a great need for early detection. Here we aim to investigate a novel signaling pathway that holds promise for early detection of triple-negative breast cancer.TASK3 is a pH sensitive potassium channel protein that regulates mitochondrial membrane potential m). Overexpression of TASK3 increases m, and thereby promotes apoptosis resistance and tolerance of hypoxia. In Preliminary Data, we show that TASK3 is overexpressed in 1) chemotherapy-resistant metastatic triple-negative breast cancers and 2) premalignant breast disease in high-risk African American women. KCNK9 (TASK3 gene) is regulated by imprinting: loss of imprinting predicts chemotherapy resistance and subsequent metastasis of triple-negative breast cancer: The gene coding for the TASK3 protein, KCNK9, is regulated by methylation imprinting. Imprinting is a normal regulatory process where one copy of the gene is inactivated resulting in mono-allelic gene expression. Loss of normal imprinting results in a functional diploid state and overexpression of the target gene. One copy of the KCNK9 gene is normally imprinted, making the normal functional state of KCNK9 haploid (one gene copy expressed, one copy not expressed). In Preliminary Data, we identified a differentially methylated region (DMR) in the KCNK9 promoter that was imprinted in normal mammary epithelial cells but not in primary chemotherapy-resistant triple-negative breast cancers. Loss of DMR imprinting predicted TASK3 overexpression, chemotherapy-resistance, and subsequent metastasis. Hypothesis: Loss of methylation imprinting of KCNK9 DMR and hypoxia synergistically promote TASK3 overexpression and metastasis in triple-negative breast cancer. Aim 1 will test whether hypoxia transcriptionally activates TASK3 in triple-negative breast cancers that lack imprinting of the KCNK9 DMR. We will perform in vivo multi-parametric analysis of KCNK9 HRE activity, TASK3 expression, tumor growth and metastasis, and hemoglobin saturation in normoxic and hypoxic conditions. Aim 2 will test whether loss of normal KCNK9 DMR imprinting promote initiation, progression, and metastasis of triple-negative breast cancer. Significance: Successful completion of the aims of this proposal will improve our ability to identify biologically aggressive metastatic triple-negative breast cancer. Triple-negative breast cancers have a high mortality in premenopausal African American women. Our established high-risk cohort will allow us to rapidly test whether loss of KCNK9 DMR imprinting promotes chemotherapy-resistant metastatic triple- negative breast cancers. PUBLIC HEALTH RELEVANCE: The molecular origins of estrogen receptor-negative (ER-) breast cancers in African American and Caucasian women are poorly understood. Importantly, we do not understand whether the molecular pathways that underlie the aggressive behavior of ER(-) breast cancers can be detected in premalignant lesions. This information is crucial for developing successful early detection and targeted-prevention strategies. Here we take trans-disciplinary approach to investigate the epigenetic origins of ER(-) breast cancer in our Duke breast cancer tissue bank and world-class prospective cohort of high-risk pre- menopausal Caucasian and African American women.. PHS 398/2590 (Rev. 09/04, Reissued 4/2006) Page Continuation Format Page